The long-term goal of this project is to understand the molecular mechanisms of hormone refractory growth and metastasis of prostate cancer (PCA). We have shown previously that posttranslational modifications, including phosphorylation and ubiquitination, play an important role in regulation of transcriptional activity and specificity of the androgen receptor (AR) under androgen-depleted conditions. The precise mechanisms by which these posttranslational modifications regulate transcription program of AR in PCA cells remain largely unknown. Our preliminary study suggest that phosphorylation and ubiquitination modifications of AR may be coupled and coordinately regulate AR activity. In addition, we have identified several novel AR splicing variants lacking the ligand-binding domain in hormone-refractory PCA cells. One of the major splicing variants, AR3, is upregulated during PCA progression and its expression level predicts the risk of tumor recurrence. AR3 may play a distinct, yet essential, role in castration-resistant growth through regulating a unique set of genes which are not regulated by the prototype AR. This has added another layer of complexity of regulation of AR-mediated transcription program. To better understand the role of AR3 in PCA development and progression, we have, for the first time, established a transgenic mouse model AR3Tg by targeted overexpression of AR3 in prostate epithelium to recapitulate pathological changes occurred in human PCA. Our initial characterization of AR3Tg revealed that enforced-expression of AR3 in prostatic epithelial cells activates multiple growth factor signaling pathways and promotes proliferation in prostate epithelium compartment, leading to an increase of cells expressing stem/progenitor cell markers. We have also showed that AR3 is phosphorylated by multiple protein kinases as well as ubiquitinated. We will utilize this animal model to study how posttranslational modifications of AR3 modulate its activity. We propose to test the hypothesis that the transcriptional activity and specificity of AR and its splicing variant AR3 may be regulated co-ordinately by multiple posttranslational modifications which may lead to differential recruitment of co-factors to the regulatory regions of their target genes. Three specific aims are proposed: Aim 1, Test whether AR transcriptional activity is coordinately regulated by phosphorylation and ubiquitination under androgen-depleted conditions; Aim 2, Examine mechanisms by which AR3 activity is regulated; Aim 3, Examine the functional interaction between AR and AR3 under both normal and Pten-deficient background and its contribution to prostate cancer progression. PUBLIC HEALTH RELEVANCE: Prostate cancer (PCA) is the most commonly diagnosed cancer among men. Androgen ablation therapy is one of the commonly used treatments for advanced prostate cancer patients. However, most patients eventually develop androgen-independent (or castration-resistant) tumors, which are highly aggressive and resistant to conventional therapies. To improve PCA therapy, it will be necessary to address the problems of progression to androgen independence. Therefore, understanding the biological mechanisms involved in androgen-independent growth has emerged as a fundamental and urgent issue in PCA research. Androgen receptor (AR) is one of the key molecules mediating androgen signaling in prostate cells. A majority of castration-resistant PCA cells express AR and androgen-responsive genes, indicating that AR-signaling pathway is still functional under androgen-depleted conditions. The mechanisms by which AR remain to be active are not well understood. In this application, we will study how posttranslational modifications modulate the activity of AR and its hormone-independent splicing variant AR3. Successful completion of the proposed research will enable us to gain new insights into molecular mechanisms underlying the development of hormone-resistance of PCA. The phosphospecific antibodies developed in this study would become potential powerful tools for diagnostic applications such as prediction of patient's response to hormonal therapy. The mouse models generated in this study, which recapitulate human disease process, would become valuable tools for further functional and therapeutic studies.